scholarly journals Adaptive laboratory evolution and reverse engineering of single-vitamin prototrophies in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Thomas Perli ◽  
Dewi P.I. Moonen ◽  
Marcel van den Broek ◽  
Jack T. Pronk ◽  
Jean-Marc Daran
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Nicotinic Acid ◽  
Specific Growth ◽  
Pantothenic Acid ◽  
B Vitamins ◽  
Fast Growth ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
B Vitamin

AbstractQuantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B-vitamins. Previous work demonstrated that, in S. cerevisiae CEN.PK113-7D, requirements for biotin could be eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA) or thiamine were omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B-vitamins. In all evolution lines, specific growth rates reached at least 90 % of the growth rate observed in SM supplemented with a complete B-vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid or pABA. Reaching similar results in SM lacking thiamine, pyridoxine or pantothenate required over 300 generations of selective growth. The genomes of evolved single-colony isolates were re-sequenced and, for each B-vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth were selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B-vitamin, introduction of a small number of mutations sufficed to achieve substantially a increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM.ImportanceMany strains of Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, nicotinic acid and inositol. However, omission of these B-vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B-vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotype by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step towards development of S. cerevisiae strains that exhibit fast growth on cheap, fully mineral media that only require complementation with a carbon source, thereby reducing costs, complexity and contamination risks in industrial yeast fermentation processes.

scholarly journals Adaptive Laboratory Evolution and Reverse Engineering of Single-Vitamin Prototrophies in Saccharomyces cerevisiae

2020 ◽  
Vol 86 (12) ◽  
Author(s):  
Thomas Perli ◽  
Dewi P. I. Moonen ◽  
Marcel van den Broek ◽  
Jack T. Pronk ◽  
Jean-Marc Daran
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Nicotinic Acid ◽  
Specific Growth ◽  
B Vitamins ◽  
Fast Growth ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Content Type ◽  
B Vitamin

ABSTRACT Quantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B vitamins. Previous work demonstrated that in S. cerevisiae CEN.PK113-7D, requirements for biotin were eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA), or thiamine was omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B vitamins. In all evolution lines, specific growth rates reached at least 90% of the growth rate observed in SM supplemented with a complete B vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid, or pABA. Reaching similar results in SM lacking thiamine, pyridoxine, or pantothenate required more than 300 generations of selective growth. The genomes of evolved single-colony isolates were resequenced, and for each B vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth was selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B vitamin, the introduction of a small number of mutations sufficed to achieve a substantially increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM. IMPORTANCE Many strains of Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, nicotinic acid, and inositol. However, omission of these B vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotypes by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step toward development of S. cerevisiae strains that exhibit fast growth on inexpensive, fully supplemented mineral media that only require complementation with a carbon source, thereby reducing costs, complexity, and contamination risks in industrial yeast fermentation processes.

scholarly journals Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Jasmine M. Bracher ◽  
Erik de Hulster ◽  
Charlotte C. Koster ◽  
Marcel van den Broek ◽  
Jean-Marc G. Daran ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Specific Growth Rate ◽  
Specific Growth ◽  
Baker’S Yeast ◽  
Industrial Biotechnology ◽  
Baker's Yeast ◽  
Laboratory Evolution ◽  
Content Type ◽  
Study Laboratory

ABSTRACT Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h−1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h−1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h−1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h−1. The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast. IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are insufficient to sustain fast growth and product formation. Consequently, this expensive vitamin is routinely added to baker's yeast cultures. In this study, laboratory evolution in biotin-free growth medium yielded new strains that grew as fast in the absence of biotin as in its presence. By analyzing the DNA sequences of evolved biotin-independent strains, mutations were identified that contributed to this ability. This work demonstrates full biotin independence of an industrially relevant yeast and identifies mutations whose introduction into other yeast strains may reduce or eliminate their biotin requirements.

scholarly journals Improving isobutanol productivity through adaptive laboratory evolution in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Aili Zhang ◽  
Yide Su ◽  
Jingzhi Li ◽  
Weiwei Zhang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Metabolic Engineering ◽  
Glucose Concentration ◽  
Evolutionary Engineering ◽  
Whole Genome ◽  
Genome Resequencing ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Whole Genome Resequencing

Abstract Background: Isobutanol is an ideal second-generation biofuels due to its lower hygroscopicity, higher energy density and higher-octane value. However, isobutanol is toxic to production organisms. To improve isobutanol productivity, adaptive laboratory evolution method was carried out to improve the tolerance of Saccharomyces cerevisiae toward higher isobutanol and higher glucose concentration.Results: We evolved the laboratory strain of S. cerevisiae W303-1A by using EMS (ethyl methanesulfonate) mutagenesis followed by adaptive laboratory evolution. The evolved strain EMS39 with significant increase in growth rate and viability in media with higher isobutanol and higher glucose concentration was obtained. Then, metabolic engineering of the evolved strain EMS39 as a platform for isobutanol production were carried out. Delta integration method was used to over-express ILV3 gene and 2μ plasmids carrying ILV2, ILV5 and ARO10 were used to over-express ILV2, ILV5 and ARO10 genes in the evolved strain EMS39 and wild type W303-1A. And the resulting strains was designated as strain EMS39V2δV3V5A10 and strain W303-1AV2δV3V5A10, respectively. Our results shown that isobutanol titers of the evolved strain EMS39 increased by 30% compared to the control strain. And isobutanol productivity of strain EMS39V2δV3V5A10 increased by 32.4% compared to strain W303-1AV2δV3V5A10. Whole genome resequencing and analysis of site-directed mutagenesis of the evolved strain EMS39 have identified important mutations. In addition, RNA-Seq-based transcriptomic analysis revealed cellular transcription profile changes resulting from EMS39.Conclusions: With the aim of increase productivity of isobutanol in S. cerevisiae, improving tolerance toward higher isobutanol and higher glucose concentration via EMS mutagenesis followed by adaptive evolutionary engineering was conducted. An evolved strain EMS39 with significant increase in growth rate and viability had been obtained. And metabolic engineering of the evolved strain as a platform for isobutanol production was carried out. Furthermore, analysis of whole genome resequencing and transcriptome sequencing were also carried out.

scholarly journals The B-vitamin content of baboon (Papio cynocephalus) milk

1977 ◽  
Vol 38 (3) ◽  
pp. 507-511 ◽  
Author(s):  
D. H. BUSS ◽  
J. E. Ford ◽  
K. J. Scott
Keyword(s):  
Dietary Intake ◽  
Nicotinic Acid ◽  
Pantothenic Acid ◽  
B Vitamins ◽  
Vitamin B ◽  
Papio Cynocephalus ◽  
Mature Milk ◽  
Vitamin Content ◽  
The Absolute ◽  
B Vitamin

1. The B-vitamins in milk from baboons (Papio cynocephalus) at various stages of lactation were measured microbiologically.2. Mature milk contained, on average (μg/ml): thiamin 0.18, riboflavin 0.74, nicotinic acid 3.2, folate 0.03, vitamin B6 0.70, vitamin B12 0.002, pantothenic acid 2.63, biotin 0.0065.3. Colostrum contained much less vitamin B6 and pantothenic acid than mature milk; otherwise, there were only slight changes in composition as lactation progressed. Neither these changes, nor the absolute values, resembled those for human or cow's milk.4. The average daily secretion of B-vitamins in milk represented less than 10% of the mother's dietary intake; that of folate was less than 0.5% of the mother's dietary intake.5. Baboon milk was calculated to provide infants with approximately the following quantities of B-vitamins (/d): thiamin 0.06 mg, riboflavin 0.25 mg, nicotinic acid 1.1 mg, folate 10 μg, vitamin B6 0.25 mg, vitamin B12 0.7 μg, pantothenic acid 0.9 mg, biotin 2.2 μg.

scholarly journals Influence of organic acids and organochlorinated insecticides on metabolism of Saccharomyces cerevisiae

2005 ◽  
pp. 207-215 ◽  
Author(s):  
Dusanka Pejin ◽  
Vesna Vasic
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Sugar Beet ◽  
Specific Growth Rate ◽  
Glycogen Content ◽  
Specific Growth ◽  
Raw Materials ◽  
Stress Factors ◽  
Adaptive Mechanism ◽  
Ribonucleic Acids

Saccharomyces cerevisiae is exposed to different stress factors during the production: osmotic, temperature, oxidative. The response to these stresses is the adaptive mechanism of cells. The raw materials Saccharomyces cerevisiae is produced from, contain metabolism products of present microorganisms and protective agents used during the growth of sugar beet for example the influence of acetic and butyric acid and organochlorinated insecticides, lindan and heptachlor, on the metabolism of Saccharomyces cerevisiae was investigated and presented in this work. The mentioned compounds affect negatively the specific growth rate, yield, content of proteins, phosphorus, total ribonucleic acids. These compounds influence the increase of trechalose and glycogen content in the Saccharomyces cerevisiae cells.

B-COMPLEX VITAMIN SUPPLEMENTATION OF PIG RATIONS BASED ON BARLEY GROWN ON GREY WOODED SOILS

1962 ◽  
Vol 42 (2) ◽  
pp. 183-190 ◽  
Author(s):  
B. D. Owen ◽  
A. Reddon ◽  
F. Whiting
Keyword(s):  
Nicotinic Acid ◽  
Post Partum ◽  
Pantothenic Acid ◽  
Vitamin Supplementation ◽  
Vitamin B ◽  
Growth Responses ◽  
Growing Period ◽  
Peace River ◽  
River Region ◽  
B Vitamin

Ninety-two sows and two hundred and sixteen pigs were employed in four gestation-lactation and three growing-finishing experiments to determine the adequacy of barley grown on the Grey Wooded soils of the Peace River region in northwestern Canada, as a source of riboflavin, pantothenic and nicotinic acid in swine rations. Vitamin B12 was assessed as a dietary essential in the all-plant rations. In the gestation-lactation experiments sow gains during gestation were greater in the B-vitamin supplemented lot and a trend toward heavier weaning weights of pigs was evident. Vitamin supplementation increased the level of vitamin B12 in sow’s milk collected 1 week post-partum and of both riboflavin and pantothenic acid collected 1 week and 6 weeks post-partum. Nicotinic acid content of sows’ milk was unaffected by supplementation. There was a highly significant decrease in riboflavin level in both control and supplemented lots between 1 and 6 weeks following parturition. The decrease was greatest in the unsupplemented lot.B-vitamin supplementation did not affect growing period gains or pig carcass quality in the growing-finishing experiments. However, certain significant growth responses were noted in the finishing period.The data indicate that the basal rations employed contained adequate riboflavin, pantothenic and nicotinic acid to support normal gestation, lactation and growth in Yorkshire swine.

scholarly journals Comparison of the Acute Postprandial Circulating B-Vitamin and Vitamer Responses to Single Breakfast Meals in Young and Older Individuals: Preliminary Secondary Outcomes of a Randomized Controlled Trial

Nutrients ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 2893 ◽  
Author(s):  
Pankaja Sharma ◽  
Nicola Gillies ◽  
Shikha Pundir ◽  
Chantal A. Pileggi ◽  
James F. Markworth ◽  
...  
Keyword(s):  
High Performance ◽  
Dietary Habits ◽  
Controlled Trial ◽  
Age Groups ◽  
Pantothenic Acid ◽  
B Vitamins ◽  
Flavin Mononucleotide ◽  
Older Individuals ◽  
Minimal Impact ◽  
B Vitamin

B-vitamin deficiency is common in ageing populations either due to altered dietary habits or altered digestive and metabolic functions. There is limited data on the acute circulating concentrations of B-vitamins and their various forms (vitamers), following ingestion of realistic meals. This study compared the acute circulating B-vitamin and vitamer responses to either an energy-dense (ED) or a nutrient-dense (ND) breakfast meal, consumed in a randomized cross-over sequence, in older and younger adults (n = 15 and 15, aged 67.3 ± 1.5 and 22.7 ± 0.5 years (mean ± SEM), respectively). Eleven differing B-vitamins and vitamers were determined in plasma samples by ultra-high-performance liquid chromatography-tandem mass spectrometry, in the fasting and postprandial state (hourly for 5 h). While postprandial thiamine concentration increased following both meals, riboflavin increased only following a ND meal in both age groups. Many vitamins including nicotinic acid, pantothenic acid, pyridoxal, pyridoxamine, pyridoxal-5’phosphate, and 4-pyridoxic acid remained unaltered, and flavin mononucleotide (FMN), nicotinamide and nicotinuric acid concentrations reduced following both meals. Biological age and food composition had minimal impact on postprandial B-vitamin concentrations, yet the differences between the ED and ND meals for riboflavin highlight the importance of riboflavin intake to achieve adequacy.

scholarly journals Intracellular symbiosis and vitamin requirements of two insects, Lasioderma serricorne and Sitodrepa panicea

1944 ◽  
Vol 132 (867) ◽  
pp. 212-221 ◽  
Keyword(s):  
Growth Rate ◽  
Nicotinic Acid ◽  
Artificial Diet ◽  
Pantothenic Acid ◽  
Lasioderma Serricorne ◽  
White Flour ◽  
Vitamin Requirements ◽  
Pure Substances ◽  
Better Than

Normal larvae of two beetles, Lasioderma serricorne and Sitodrepa panicea , and larvae freed from their intracellular symbionts have been grown on various diets. On white flour, a food deficient in vitamins of the B group, normal larvae grow very much better than sterilized larvae, while no such difference in the growth rate occurs on a diet rich in vitamins of the B group, e. g. wholemeal flour plus yeast. On an artificial diet which contains the vitamins of the B complex in pure substances, the normal Lasioderma larva grows well or fairly well in the absence of either thiamin, riboflavin, nicotinic acid, pyridoxin or pantothenic acid, and the normal Sitodrepa larva in the absence of any of these vitamins except thiamin. The sterilized larvae of both species fail to grow in the absence of any of these five vitamins. It is concluded that the intracellular symbionts of Lasioderma and Sitodrepa supply vitamins of the B group.

scholarly journals Anaplerotic reactions active during growth of Saccharomyces cerevisiae on glycerol

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Joeline Xiberras ◽  
Mathias Klein ◽  
Celina Prosch ◽  
Zahabiya Malubhoy ◽  
Elke Nevoigt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Specific Growth Rate ◽  
Pyruvate Carboxylase ◽  
Reference Strain ◽  
Specific Growth ◽  
Glyoxylate Cycle ◽  
Maximum Specific Growth Rate ◽  
The Glyoxylate Cycle ◽  
Anaplerotic Reactions

ABSTRACT Anaplerotic reactions replenish TCA cycle intermediates during growth. In Saccharomyces cerevisiae, pyruvate carboxylase and the glyoxylate cycle have been experimentally identified to be the main anaplerotic routes during growth on glucose (C6) and ethanol (C2), respectively. The current study investigates the importance of the two isoenzymes of pyruvate carboxylase (PYC1 and PYC2) and one of the key enzymes of the glyoxylate cycle (ICL1) for growth on glycerol (C3) as a sole carbon source. As the wild-type strains of the CEN.PK family are unable to grow in pure synthetic glycerol medium, a reverse engineered derivative showing a maximum specific growth rate of 0.14 h−1 was used as the reference strain. While the deletion of PYC1 reduced the maximum specific growth rate by about 38%, the deletion of PYC2 had no significant impact, neither in the reference strain nor in the pyc1Δ mutant. The deletion of ICL1 only marginally reduced growth of the reference strain but further decreased the growth rate of the pyc1 deletion strain by 20%. Interestingly, the triple deletion (pyc1Δ pyc2Δ icl1Δ) did not show any growth. Therefore, both the pyruvate carboxylase and the glyoxylate cycle are involved in anaplerosis during growth on glycerol.

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